JPH11504521A - Plant pathogen resistance gene and use thereof - Google Patents

Abstract

(57) Abstract: The tomato Cf-4 gene is isolated by positional cloning and its sequence is provided along with the encoded amino acid sequence. DNA encoding a polypeptide, alleles, variants and derivatives thereof, and DNA encoding an amino acid sequence exhibiting a significant degree of homology thereto are introduced into plant cells, and the encoded polypeptide is expressed. Pathogen resistance can be conferred on plants comprising such cells and their progeny. The Cf-4 sequence shows a high degree of homology to Cf-9 and comprises a high leucine repeat.

Description

DETAILED DESCRIPTION OF THE INVENTION Plant pathogen resistance gene and use thereof   The present invention relates to pathogen resistance in plants, and more particularly, to the pathogen resistance gene For identification and use. It is a tomatoCf-4Based on gene cloning.   Plants are constantly attacked by potentially pathogenic microorganisms. Crops are usually left Because they are grown as a monoculture that is genetically uniform, Vulnerable to harm. However, plants have both preexisting and induced defense lines. Have been evolved, but due to their close association with pathogens, especially living plant cells Pathogens that gain their nutrition must evade their defenses. Pathogen If a plant can cause disease, such an interaction is said to be compatible and If an object is resistant, the interaction is said to be incompatible.   Breed-specific resistance is often specified (but not exclusively) by the dominant R gene. Mutations are often recessive when the pathogen mutates to overcome the R gene . In order for the R gene to function, it is called an avirulence gene (Avr). The corresponding gene in the pathogen must also be present. To become toxic, the pathogen The body (fungi, bacteria or viruses) no longer triggers R gene-dependent defense mechanisms The substance must be produced (Flor, 1971). Often elicitor / One model, called the receptor model, is based on the Avr gene So that the R gene can detect the presence of the pathogen in plants (See Gabriel and Rolfe, 1990). So After this Knowledge is translated into activation of defense responses.   Cladosporium fulvum, a tomato humus pathogenCladosporium fulvu m ) Have been reported.Avr9WhenAvr4 The gene encodes a small high cysteine peptide (van Kan et al., 1991; Josten). Et al., 1995), and in their processed mature forms, both genes are 28 and 28 respectively. And 106 amino acid residues.Avr9WhenAvr4Is the R geneCf-9WhenCf -Four Is added to the tomato line carrying Confers non-pathogenicity to fulvum varieties . We have determined whether the two R genes are genetically closely linked, Have shown that they may be alleles (Jones et al., 1993; Ba lint-Kurti et al., 1994). The present inventors have proposed a transposon labeling method.Cf-9Gene Of isolated (Jones et al., 1994; PCT / GB94 / 02812 published as WO 95/18230) In this specification, as a more sophisticated gene analysis and probeCf-9Using DNA WasCf-4We report the position cloning of. Cloned non-toxic genes and their The availability of these same resistance genes will ultimately be widespread in a wide range of crops. It can be used to build type and permanent disease resistance (de Wit, 1992; Staskawicz et al., 1995).   In plants, gene isolation has two main approaches: positional cloning and Achieved by transposon labeling. Some plant genes are Successfully isolated by tanning (reviewed in Tanksley et al., 1995). ), Some of those genesRGene, iePto(Martin et al., 1993) And from tomatoesCf-2(Dixon et al., 1996) and Brassicaceae (Arabidopsis )fromRPS2andRPM1(Bent et al., 1994; Mindrinos et al., 1994; Grant et al., 1995) Is mentioned. Most positional cloning methods have limitations It relies heavily on the use of fragment length polymorphism (RFLP) markers. However, most Closer, allowing more DNA sequences to be investigated for polymorphism, A PCR-based method that can detect more subtle DNA sequence variations is It has been developed. These techniques are based on random amplified polymorphic DNA (RAPD, Williams et al., 19). 90) and amplification restriction fragment polymorphism analysis (AFLP, Zabeau and Vos, 1992, EP-A-9240) Vos et al., 1995; Thomas et al., 1995) and positional cloning methods. Will facilitate the isolation of plant genes. Transposon labeling with tobacco FromNGene (Whitham et al., 1994) from flaxL ⁶ (Lawrence et al., 1995), Thomas FromCf-9(Jones et al., 1994; PCT / GB94 / 02812 published as WO 95/18230) Used to isolate   plantRComparison of the amino acid sequences of genes indicates that at present they This is shown in FIG. All but PTO have high leucine repeat (LRR) Seems to contain motifs, butRGeneticL ⁶,N,RPM1andRPS2IsCf- 9 (Staskawicz et al., 1995) andCf-2(Dixon et al., 1996) It seems to have areas. Furthermore,L ⁶,NandRPS2Consists mostly of LRR And is expected to be mainly a cytoplasmic outer membrane-bound proteinCf-9andCf-2What is In contrast, it will probably be intracellular. Our analysis shows a presumedCf-4 ProteinCf-9Indicates that it is highly homologous to   WO 93/11241 isCf-9And we just discoveredCf-4(Subject of the present invention Polygalacturonase inhibitory protein (PGIP) with some homology to The sequence of the gene encoding is reported.Cf-9,Cf-4And others (Cf-5,-2 Are referred to by those skilled in the art as "pathogen resistance genes" or "disease resistance genes." Have been. The gene encoding PGIP is not a pathogen resistance gene. Pathogen resistance gene (R) Is the corresponding non-pathogenic gene (AvrPlants detect presence of pathogens that express It can be so. When a pathogen is detected, defenses such as hypersensitivity reactions (HR) The response is activated. By such means, the plant is located at the site where the pathogen attempted to invade. Local cell death can kill pathogens in living cells. On the other hand PGIP gene (eg, WO 93/11241) results from the detection of pathogens by the R gene This is a type of gene that is induced in plant defense responses.   Therefore, the pathogen resistance gene is derived from the pathogen andAvrDependent molecule It can be considered as encoding a condition. Thus, it is PGIP was detected once, whereas it can be compared to the plant RADAR for Are involved in plant-initiated defense against pathogens and in pathogen resistance genes Absent. Expression of a pathogen resistance gene in a plant activates defense responses in that plant cause. This is when the plant contacts the pathogen or the corresponding elicitor molecule. Can result in overexpression of the resistance gene in the absence of elicitor. The possibility of causing more activation has been reported. The defense response is local, even if Activated at the site of contact between the plant and the pathogen or elicitor molecule or systemically It may be. Activation of defense responses in plants expressing pathogen resistance genes is not appropriate. The corresponding elicitor molecule, eg Cladosporium fur as described Bam (Cladosporium fulvum)avrThings produced by genes, and plants It can also be caused by contact with Elicitor Cladosporium Lubham (Cladosporium fulvum) In pathogen extracts such as May be wholly or partially purified and fully or partially synthesized. May be used. The elicitor molecule causes it to activate a defense response If the appropriate ligand for the R gene product to Can be said.   The terms "Cf-x" / "Avrx" are standard in the art.CfResistance gene and The corresponding fungal non-pathogenic gene (Avr) Initially has its measurable activity Genetically defined as an interacting pair of genes belonging to an exclusive interacting pair Was. The components recognized by Cf-4 are different from those recognized by Cf-9 so,Avr9IsCf-9Induces a necrotic response to contained tomatoes,Cf-4Containing tomatoes Does not elicit a response at all.   Expression of Cf-4 function in plants is dependent on various C. Investigating compatibility of fulbum varieties Can be determined by   All knownAvrC. having a functional copy of the gene Variety of fulbum (variety 0) Is allCfWill grow only on tomatoes that lack the gene (is compatible). It has any functionalityCfDoes not grow on plants carrying the gene (incompatible ). If C. Fulbum variety is functionalAvr4If you lack the gene Species 4), it is eitherCfNot only can they grow on plants that lack genes ,Cf-4It could also grow on plants with the gene. FunctionalityAvr2Lacking genes The varieties (varieties 2 and 4)Cf-2It could grow on plants with the gene. Machine AbilityAvr2Varieties lacking only genes (variety 2)Cf-4Propagation on plants with genes I can't. Similarly, C.I. Fulbum variety 5 (functionalAvr5Lacking the gene)Cf-4 Will not be able to grow on plants with the gene. Both varieties 4 and varieties 2 and 4 Even otherCfWill not be able to grow on plants with any of the genes. Various varieties Are commonly available in the art, for example, the Research Institute for Plant Pro tection (IPO-DLO), PO Box 9060, 6700 Available from GW Wageningen, The Netherlands. Variety 4 has accession number IPO103 Available under 79 and varieties 2 and 4 are available under accession number IPO50379 It is possible.   Cf-2The gene was provided by John Innes Center Innovations Limited on April 1, 1996. PCT / GB96 / 00785 subject to application and claiming priority from GB9506658.5 It is.   The present inventors have now found that the fungus Cladosporium fulvum (Cladosporium fulvum Tomato gene conferring resistance to)Cf-4And its DNA The sequence and deduced amino acid sequence were determined. TomatoCf-4Figure 5 shows the DNA sequence of the gene. (SEQ ID NO: 1) and the deduced amino acid sequence is shown in FIG. 6 (SEQ ID NO: 2).   In one aspect, the invention relates to a nucleic acid isolate encoding a pathogen resistance gene. The gene is the amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof; It encodes an amino acid sequence showing a considerable degree of homology to A nucleic acid isolate. this is,Cf-9,Cf-2andCf-5One of the nets No greater than the amino acid sequence of SEQ ID NO: 2 Column identity can be. This can be at least about 95% identity Wear.   Most preferably, the nucleic acid encodes the amino acid sequence shown in SEQ ID NO: 2, In this case, the nucleic acid isolate is a nucleic acid having the sequence shown in SEQ ID NO: 1, or A portion sufficient to encode the desired polypeptide (eg, a methionine initiation codon or To the first downstream stop codon in the frame). One aspect A nucleotide sequence wherein the DNA is nucleotides 201 to 2618 of SEQ ID NO: 1; Or a variant, derivative or allele thereof.   A further aspect of the invention relates to nucleotides 201 to 2618 of SEQ ID NO: 1 or Uses probes comprising fragments, derivatives, variants or alleles thereof. To screen the DNA library for pathogen resistance in plants, e.g. Varieties of Radosporium fulvum (egAvr4Varieties expressing) By isolating DNA encoding a polypeptide that can be conferred. Provided, a nucleic acid isolate encoding a pathogen resistance gene, or a fragment thereof . The plant can be a tomato. Suitable techniques are well known in the art.   Thus, the present invention identifies nucleic acids encoding pathogen resistance genes and / or Is a method of isolating, encoding the amino acid sequence shown in SEQ ID NO: 2, Complementary to, or complementary to, coding sequence Using a nucleic acid having a nucleotide sequence that encodes a fragment of any of the following sequences: Also provided is a method comprising probing a candidate (or "target") nucleic acid. The candidate nucleic acid (which can be, for example, cDNA or genomic DNA) is Contains, or is suspected of containing, a nucleic acid encoding a pathogen resistance gene It can be obtained from any cell or organism. The preferred nucleotide sequence is SEQ ID NO: No. 1. Sequences complementary to the indicated sequence, and fragments thereof, may be used .   Preferred search conditions include a small number of positively identified Strain enough to obtain a simple pattern with hybridization Is a condition. Hybridization until only a few positive clones remain It is well known in the art to gradually increase the stringency of DNA.   The nucleic acid of the present invention has the amino acid sequence shown in SEQ ID NO: 2 or the given sequence Can encode a variant, derivative or allele of Preferred mutation Body, derivative and allele Confer functional characteristics of the protein encoded by the gene, especially pathogen resistance Ability, most particularlyAvr4Confers resistance to pathogens expressing elicitor molecules Ability. Sequences for creating variants or derivatives Alteration results in an insertion, deletion or substitution of one or more amino acids. One or more insertions, deletions or substitutions of one or more nucleotides in a nucleic acid Or it can be multiple. Of course, the encoded amino acid sequence Nucleic acid changes that do not result in a change are also included.   According to one aspect of the present invention, any of the coding sequences provided herein may A nucleus comprising a nucleotide sequence complementary to the hybridizable nucleotide sequence An acid is also provided. Another aspect of this is that nucleic acids in this regard are provided herein. Hybridizable to a nucleotide sequence complementary to any of the coding sequences It is. Of course, DNA is usually double-stranded, and after strand separation, which of the strands is high Southern hybridizations with or without distinction A blotting technique, such as an alternative, is performed. Preferably, a hybridizable nucleus The acid or its complement is a polypeptide that can confer pathogen resistance to the host. And thus contains a pathogen resistance gene. Preferred hybridisation Although the condition of the arrangement is well known to those skilled in the art, it is usually noted that the other conditions are excluded. Stringency is sufficient to allow positive hybridization between the sequences.   Nucleic acids of the invention, e.g., variants, derivatives and derivatives of the specific sequences disclosed herein. And alleles may be due to one or more of the following characteristics, or Other features that can be directly derived from the comparison of the amino acid sequence to each other sequence, Is based on the observation of gene functionCf-9,Cf-2And / orCf-5Can be distinguished from it can: The amino acid sequence has at least about 95% identity to the amino acid sequence of SEQ ID NO: 2 Have; -For example, Cladosporium fulvum expressing Avr4 (Cladosporium fulvum A) Avr4 elicitor as given by variety (available in the state of the art) When a molecule is brought into contact with a plant, a defense response is induced in a plant that expresses the nucleic acid To do; − Research Institute for Plant Protection (IPO-DLO), PO Box 9060, 6700 Deposited with GW Wageningen, The Netherlands under accession number IPO10379 And C. available from there. Fulbam cultivar 4 or its extract and plant Does not elicit a protective response in plants expressing the nucleic acid when contacted; -Deposited with and available from the institution under accession number IPO50379 C. When the plant is brought into contact with fulbum varieties 2, 4 or an extract thereof, Not elicit a protective response in plants expressing the nucleic acid; -For example, by A. cerevisiae cultivars or other organisms expressing Avr4. Avr4 elicitor molecule as provided in SEQ ID NO: 13 of WO 95/31564 and the book The amino acid sequence given in FIG. 4 of the specification and the nucleic acid sequence Elicit a protective response in a plant that expresses the nucleic acid when contacted with a substance ; -For example, by a Cladosporium fulvum cultivar or other organism expressing Avr9. Avr9 elicitor molecule as provided (de Wit, 1992), eg, WO 95/18230 Texture as given in SEQ ID NO: 3 and in WO 95/31564 as SEQ ID NO: 4 The plant was brought into contact with the amino acid sequence of la form and the nucleic acid sequence encoding it Sometimes not elicit a protective response in plants expressing said nucleic acid; -For example, Cladosporium fulvum varieties expressing Avr2 or When the plant is brought into contact with the Avr2 elicitor molecule as provided by other organisms Not elicit a protective response in plants expressing the nucleic acid; -For example, by A. cerevisiae cultivars or other organisms expressing Avr5. When the plant is brought into contact with the Avr5 elicitor molecule as provided, the nucleic acid is released. Not elicit a protective response in the emerging plant; -In a plant expressing said nucleic acid and the corresponding elicitor molecule,Cf-9gene And faster than the defense responses elicited in plants expressing the corresponding Avr9 molecule Eliciting a protective response during development; -Contacting a corresponding elicitor molecule, for example Avr4, with a plant, Eliciting a protective response in non-photosynthetic tissues (eg roots) of the expressing plant; -Can be identified from the sequence information provided herein for Cf-4 Comprising the number of high leucine repeat sequences (LRR).   Cf-4 polypeptides, and others like Cf-9, Cf-2 and Cf-5, are putative membranes. Differentiate from products of other pathogen resistance genes by being penetrant proteins Can be For example, cruciferous (Arabidopsis)RPP5Gene products of genes Is a putative cytoplasmic protein.   The nucleic acid isolate of the present invention is characterized in that its expression in a plant has a protective response activity in the plant. Encoding a pathogen resistance gene capable of causing A nucleotide sequence encoding a polypeptide comprising the amino acid sequence Can consist of   Such activation can result in contact between the pathogen or the corresponding elicitor molecule and the plant. I can tell you.   Said nucleotide sequence comprises the coding sequence shown in SEQ ID NO: 1. Can consist of For example, the sequence comprises nucleotides 201 to 2619 of SEQ ID NO: 1. Can comprise.   The nucleic acid can be used for addition, insertion, deletion or substitution of one or more nucleotides. Alleles, derivatives or variants of the coding sequence shown in SEQ ID NO: 1. Can comprise a nucleotide sequence consisting of:   The nucleic acid of the present invention is characterized in that its expression in a plant triggers the activation of a defense response in the plant. A pathogen resistance gene which can be raised and which encodes a polypeptide. A polypeptide comprising one or more amino acids. Allelic variation of the amino acid sequence shown in SEQ ID NO: 2 due to addition, insertion, deletion or substitution. A pathogen-resistant residue comprising an amino acid sequence comprising a gene, derivative or variant You can code a gene.   SEQ ID NO: 2 or an amino acid sequence exhibiting a considerable degree of homology thereto. Contains DNA encoding the amino acid sequence as genomic DNA or as cDNA A nucleic acid isolate of the invention that can be used is a recombinant vector, such as a phage or It can be in the form of a sumid vector. The DNA can be expressed in host cells (eg, plant cells). Vesicles) under the control of appropriate promoters and regulatory elements Good. In the case of genomic DNA, this includes its own promoter and regulatory elements In the case of genomic DNA, this may be a suitable promoter for expression in the host cell. And regulatory factors.   One of skill in the art will prepare vectors for recombinant gene expression and develop protocols. There is enough ability to devise. Appropriate regulatory sequences, such as promoter sequences, tags Terminator fragment, polyadenylation sequence, enhancer sequence, marker gene and And other sequences as appropriate. The appropriate vector can be selected or made. For more details, see the example For example,Molecular Cloning: A Laboratory Manual, Second Edition, Sambrook et al., 1989, See Cold Spring Harbor Laboratory Press. Preparation of nucleic acid constructs, for example Used for mutagenesis, sequencing, introduction of DNA into cells and gene expression Numerous known techniques for the engineering of nucleic acids used and for the analysis of proteins and The protocol isShort Protocols in Molecular Biology, 2nd edition, Ausubel, etc. Ed., John Wiley & Sons, 1992. Sambrook et al. And A The disclosures of usubel et al., as well as all other references cited herein, are incorporated by reference. Incorporated herein.   The nucleic acid molecules and vectors of the present invention, in substantially pure or homogeneous form, Of any origin other than the sequence encoding the polypeptide having the necessary function. Is free or substantially free of the nucleic acid or gene of the species of interest. Can be provided isolated and / or purified from their natural environment. You. The nucleic acids of the invention can comprise cDNA, RNA, genomic DNA, And can be wholly or partially synthetic. "Isolate" The term encompasses all of those possibilities.   When introducing a given gene construct into a cell, several considerations well known to those skilled in the art Things have to be taken into account. Efficiency to promote transcription of nucleic acid to be inserted Can be constructed in a construct that includes a specific regulatory element. Introducing the construct into cells The method of entry must be available. Once the components have entered the cell membrane The integration into endogenous chromosomal material may occur according to various aspects of the invention. Or it need not happen. Finally, as far as plants are concerned, the target cell type is It should be able to regenerate cells into whole plants.   The plant transformed with the DNA segment containing the presequence contains the plant gene. It can be made by standard techniques already known for operation. any Appropriate technology, such as Agrobacterium (Agro bacterium ) Proprietary disarmed Ti plasmid vector (EP-A-270355, EP-A-011671) 8, Bevan, 1984), particle or microprojectile impact (US 5100792, EP-A-444882, EP-A-434616), microinjection (WO 92/09696, WO 94) / 00583, EP 331083, EP 175966), electroporation (EP 290395, WO 87) 06614) or other forms of direct DNA uptake (DE 4005152, WO 9012096, US 46) 84611) can be used to transform plant cells with DNA. A Clobacterium transformation is well known to those skilled in the art for transforming dicotyledonous species. It is widely used. Agrobacterium has some monocots due to foreign DNA It has been reported that plant species can be transformed (WO 92/14828) However, if Agrobacterium is inefficient or ineffective, Preferred for projectile bombardment, electroporation or direct DNA uptake Good. Alternatively, using a combination of different technologies, for example, Agrobacterium Impact with coated microparticles (EP-A-486234) or Culture with Agrobacterium after microprojectile bombardment (EP -A-486233) can be used to increase the efficiency of the transformation process.   The particular choice of transformation technique depends on its efficiency in transforming a plant host, as well as its efficiency. Will depend on the experience and preferences of those practicing the invention using the particular method chosen. Will be determined. The particular choice of transformation method for introducing nucleic acids into plant cells is It is not important to the present invention and does not limit the present invention.   Cf-4Gene and its variants, for exampleCf-4Genes, their alleles, variants And derivatives that show a significant degree of homology to the protein product of the derivative. What is added to plants, especially tomatoes, C.I. Resistant to pathogens such as fulvum It can be used to impart properties. For this example,Cf-4Same dye as gene Tomato genus having a color body position [Lycopersicum hirsutum (Lycopersicon hi rsutum )] Or any subcloned fragment thereof. I can do it. For this purpose, the above-mentioned vectors are transformed into live transgenic plants. Can be used for birth. Such plants areCf-4Pathogen resistance conferred by genes Property.   The invention further relates to host cells, especially plants, transformed with such vectors. Or microbial cells. Thus, a host cell comprising a nucleic acid of the invention, e.g. For example, a plant cell is provided. In a cell, the nucleic acid may be integrated into a chromosome .   Vectors comprising the nucleic acids of the invention are particularly useful for recombination into the genome. If the vector is to be used to introduce the vector into cells, the promoter Need not be included.   According to the present invention, a nucleotide sequence as provided by the present invention is encoded by Under the operable control of a promoter for controlling the expression of the polypeptide Plant cells that integrate into their genomes are also provided. Another aspect of the invention relates to plants Including introducing a vector comprising the nucleotide sequence into the cell. And a method for producing such plant cells. After such introduction, the vector and plant cell The nucleotide sequence can be introduced into the genome by recombination with the genome. Can be. Next, the polypeptide encoded by the introduced nucleic acid is expressed. Can be closed.   Plants comprising the plant cells of the present invention, as well as the Clone, seed, inbred or hybrid progeny and progeny, and any of them Also provided are optional parts of the plant, such as cuttings and seeds. The present invention relates to any plant nutrient Body, any part that can be used for sexual or asexual reproduction or regeneration (insertion (Including trees, seeds, etc.).   The present invention further provides an early stage of introducing nucleic acids into cells of a plant or its ancestors. Later, in a plant cell, the amino acid sequence of SEQ ID NO: 2, or a variant or allele thereof From genes or derivatives, or nucleic acids encoding significantly homologous amino acid sequences Comprising the expression of (producing the polypeptide encoded thereby) Provide the law. Such a method can confer pathogen resistance on the plant. It is published in WO 91/15585 (Mogen) or more preferably as WO 95/31564 According to any of the methods described in PCT / GB95 / 01075,Avr4Genes and Or any other gene involved in conferring pathogen resistance Can be.   Cf-4,Cf-9andCf-2The genes are as follows: Proliferation of fulvum It works in the same way in giving the tomatoes resistance to prevention. However And they are differentAvrMove by recognizing products, they stop the pathogen from growing There are subtle differences in the rate of stimulating the tolerance response (Hammond-Kosack and Jones 1994; Ashfield et al., 1994). The differences are described herein and in WO 95/31564 (supra). It can be used to its fullest extent for the applications indicated.   Genes that are stably integrated into the genome of a plant are transferred from generation to generation of the plant. Inherited by the grandchild, the progeny's cells may express the encoded polypeptide. And thus can have enhanced pathogen resistance. Pathogen resistance is a disease By assessing the compatibility of the drug substance (eg Cladosporium fulvum), Or non-pathogen Virulence genes (eg,Avr-4) Or for example as described herein. In the form of a recombinant virusAvr-4By using gene product transmission, Can be determined.   Cf-4Gene sequence analysisCf-9Genes (PCT / GB94 / 02812; Jones et al., 1994)Cf-2 Like the gene (PCT / GB96 / 00785; Dixon et al., 1996), it is Containing the DNA sequence encoding the repeat (LRR) region, and homology studies Revealed strong homology with other genes, including LRR. It All three of these genes show similar general characteristics, and as such have been characterized to date. A new class of disease resistance genes that is different from other disease resistance genes that have been identified.   According to a further aspect, the present invention provides a method for searching for a novel resistance gene, comprising the steps of: Between children, for exampleCf-9WhenCf-4Between and / orCf-4WhenCf-2Saved between A plant comprising the use of an oligonucleotide comprising one or more sequences Methods for identifying pathogen resistance genes are provided. Therefore, including the pathogen resistance gene Consisting of a nucleic acid encoding a polypeptide capable of conferring pathogen resistance A method of obtaining an oligonucleotide (the details of which are described herein). Or a nucleic acid molecule comprising such an oligonucleotide as a target / candidate nucleic acid. A method is provided that comprises hybridizing. Target or candidate nucleus Acids can be obtained, for example, from organisms known to encode pathogen resistance genes. Genomic or cDNA libraries. Hybrida Clones to identify and target for further investigation and / or use / Candidate candidate nucleic acids.   Hybridization probes for nucleic acids under appropriately stringent conditions and Identify hybridization (according to known techniques) And / or as a primer in a nucleic acid amplification method (eg, PCR) It may require the use of oligonucleotides. Preferred conditions for exploration are A small number of hybridizations identified as positive so that they can be investigated These conditions are stringent enough to obtain a simple pattern with the option. Increase the stringency of hybridization until only a few positive clones remain. Is well known in the art.   Although still using nucleic acid hybridization as an alternative to exploration, Oligonucleotides designed to amplify DNA sequences Protocol in a PCR reaction or in a method involving another nucleic acid amplification. Can be used. For example, see “PCR Protocols: A Guide to Methods and Applic ations ", Innis et al., 1990, Academic Press, New York.   Appropriate preferred amino acids used in probe or PCR primer design Noic acid sequences are conserved among polypeptides that can confer pathogen resistance ( Fully, substantially or partially) sequences, egCf-4WhenCf-9By and / or OrCf-4WhenCf-2And / orCf-4,Cf-9andCf-2Coded by Array.   Based on the amino acid sequence information, the degeneracy of the genetic code and, where appropriate, the candidate nucleic acid Oligonucleotides, taking into account the codon usage of the organism from which the Probes or primers can be designed.   Preferably, for example, the oligonucleotide of the present invention used for nucleic acid amplification has about 10 Have the following codons (e.g., 6, 7, or 8), i.e., Otides (eg 18, 21, or 24).   Evaluation of whether a PCR product corresponds to a resistance gene should be performed in various ways Can be. PCR bands contain a complex mixture of products Can be Clone the individual products and add each to this probe Linkage to a known disease resistance gene segregating in progeny that showed polymorphism Can be screened for. Alternatively, the PCR product is Processed to show polymorphism on acrylamide DNA sequencing gel And preselection of a specific band associated with the resistance gene before cloning. Can be Once a candidate PCR band has been cloned and has a known resistance Once linked to a gene, it can be used to isolate clones and About other features andCf-9,Cf-2,Cf-4Or homology with other related genes You can find out more. Then analyze it by transformation and pay attention Can be evaluated for its ability to introduce plants into disease-susceptible varieties. Alternatively, using a PCR band or a sequence derived by analyzing it Can help plant breeders monitor the isolation of useful resistance genes it can.   These techniques are generally applicable to the identification of plant pathogen resistance genes. Examples of the types of genes that can be identified by this method include potato epidemics Disease fungus Phytophthora (Phytophthora) Resistant, grain spores like barley and corn Mildew and rust resistance, snapdragon (Antirrhinum) And flax rust Tolerance, lettuce and cruciferous (ArabidopsisDowny mildew resistance, potatoes, tomatoes Virus resistance in tobacco and tobacco, nematode resistance in tomatoes, bacterial disease in crucifers and tomatoes Resistance to the drug substance, as well as the pepper Zantomonas (Xanthomonas) Resistance I can do it.   Once the pathogen resistance gene has been identified, it is planted using techniques well known to those skilled in the art. The transgenic plant can be produced by re-introduction into a target cell. Further According to one aspect, the present invention relates to the presence of an LRR. Or especially the protein of the plant pathogen gene identified by the techniques described above. A DNA isolate encoding a quality product is provided.   According to yet another aspect, the invention relates to the presence of an LRR as disclosed herein. Or pathogen resistance genes identified by nucleic acid hybridization Providing transgenic plants, especially crops, which have been genetically engineered to have You.   Examples of plants according to the present invention include tobacco, cucurbitaceae, carrots, cruciferous plants , Lettuce, strawberry, cruciferous oilseed, sugarcane, wheat, barley, corn Seeds, rice, soybeans, peas, sorghum, sunflowers, tomatoes, potatoes, tows Capsicum, chrysanthemum, carnation, poplar, eucalyptus and pine.   Variations of the invention, as well as other aspects and embodiments, will be apparent to those skilled in the art. This specification All documents cited therein are incorporated by reference. The term "comprises" , Meaning "contains" or "has" and means "consists of" Should not be interpreted.   As already pointed out, the present invention relates to tomatoCf-4For gene cloning and sequencing This experimental procedure is described in more detail below with reference to the following drawings. explain.   FIG.IsCf-9(Cf9) orCf-4(Cf4) Tomato containing any of the resistance genes (L . e sculentum cv. Moneymaker) (NIL; near isogenic li ne), C.I. Strains that do not contain a known resistance gene to fulbum (Cf0), and Also mapped to the short arm of mat chromosome 1Cf-1Another containing the resistance gene Line (Cf1,L . esculentum cv. Stirling Castle)BglTurn off with II 3 shows a Southern blot of the converted DNA fragment.Cf-96.7 kbp corresponding to the geneB gl II band, andCf-4Specific to the containing strain and described in the text of the specification ThreeBglThe position of the II band is also indicated.   FIG.IsCf-4/Cf-9Five disease susceptibilities from a trans-heterozygous test cross population Three classes identified by analysis of recombinants (V408, V512, V514, V516 and V517) Recombinants are shown in schematic form.Cf-4Loci on the top rowCf-9Loci on the bottom row Is depicted.Cf-9Two AFLP markers adjacent to the gene (M1 and M2, Thomas et al. , 1995). One of those markers (M2)Cf-4Heredity It also exists in Koza. Present in several disease-susceptible recombinants,Cf-9Distal to Another two members of the multigene family located at Also shown in the figure as genes A and B). Southern hybridization Recombination breakpoints in each of the three classes estimated by Represented by a line.   FIG.IsCf-4/Cf-5Three overlapping cosmids isolated from a cosmid library (4 / 5-108, 4 / 5-129 and 4 / 5-138)Cf-4Physical map of the locus Is shown. The physical extent of each cosmid is schematically shown and the hybridization And the sequence analysisCf-9Position of homologous sequence in black box Is represented by The transcription polarity of each gene is indicated by an arrow.Cf-4Gene location Is shown. Restriction sites for the following enzymes are also shown:BglII,PSTI,EcoRI and XbaI.Cf-9Shows high homology to probes derived from the 5 'end of ThreeBglThe positions of the II fragments (6.4 kbp, 3.2 kbp and 1.8 kbp) (see FIG. 1) are indicated by arrows. Indicated by marks. M2 is the beginningCf-9Identified at the locusCf-4At the locus Represents an existing AFLP marker.   FIG.Is C.I. Proposed to code post-processing mature form of Fulbum's AVR4 Encoding the PR1a signal peptide sequence fused to the sequenceClaI /SalI DN Presumed to be the nucleic acid (double-stranded form) sequence of fragment A 1 shows an amino acid sequence. The translation start codon is at nucleotide 5 and the translation stop codon The don begins at nucleotide 413. Amino acids 1 to 30 are signal peptides In the sequence, amino acids 31-136 represent the mature AVR4 peptide.   FIG.IsCf-41 shows the genomic DNA sequence (SEQ ID NO: 1). The features of the sequence are as follows: Including: translation initiation site at nucleotide 201; beginning at nucleotide 2619 Entire translation termination; consensus polyadenylation sequence beginning at nucleotide 2835 (AATAAA); A splice donor sequence in the 3 'untranslated sequence at nucleotide 2641; Splice acceptor sequence ending in Otide 2755; putative at nucleotide 2955 Polyadenylation site.   FIG.Is a single letter amino acid symbolCf-4Deduced amino acid sequence of the gene (SEQ ID NO: 2) is shown. The deduced protein sequence consists of the primary translation product of 806 amino acids. Signal peptide amino acids 1-23; mature peptide amino acids 24-806 .   FIG.By Jones et al. (1994)Cf-9Described and discussed in the text. 7 shows the seven proposed structural domains AG of the Cf-4 protein as shown.Cf-9 Compared toCf-4The deletion in the middle is indicated by a dot. Distinguish between two proteins Most of the amino acids that are located at the N-terminus of each of them It is shown in font. Potential N-glycosylation sequences are underlined.   FIG.Indicates two major regions of sequence difference between the Cf-4 and Cf-9 sequences. (A) Do Between main Cf-9 amino acids 40-79 (top row) and Cf-4 amino acids 40-69 (bottom row) Alignment. (B) Cf-9 amino acids 285-426 (LRRs 9-14) and Cf-4 amino acids 274-369 (LRR 9-12).   FIG.Is widely used for transgene expression as reported herein. 4 shows the structures of four binary vector plasmids obtained. LB and RB are These correspond to the left and right ends of the T-DNA in each vector. Plant transformation marker The gene is located at the LB end and the neomycin phosphotransferase gene Conferring resistance to offspring (NPT) or phosphinothricin Seth Hygroscopicus (Streptomyces hygroscopicus) -Derived gene (BAR )Met. Transformation marker gene is cauliflower mosaic virus 35S pro Motor (35S) or A. Claws fascence (A. tumefaciensNopa of origin) It was placed under the control of the phosphorus synthase gene promoter (pnos). Translation termination sig Naru is A. 3 ′ sequence of octopine synthase gene of Tumefaciens (ocs3 ′) Provided by SLJ7291, SLJ7292 and SLJ755A are all indicated in the drawing. A polylinker sequence having a restriction enzyme site. This polylinker sequence is obtained from the modified pBluescript plasmid described by nes et al. (1992). all Is indicated by an arrow. All operations are described by Jones et al. (1992) Performed as described. SLJ10080 isClaLinearization with I, T4 DNA polymerase Processing and self-ligation in the polylinker sequenceClaBy removing the I site Derived from the binary cosmid vector pCLD04541. 35S from SLJ4K1:uidACassetteEcoRI /BamOf the plasmid produced as an HI fragment Cloned into. For transient expression experimentsCf-4andCf-9Code arrayClaI /BamCloned as HI fragmentuidAThe gene was replaced (see FIG. 10). Step Rasmid SLJ755A and pCLD04541 and their derivatives are Contains the lambda phage sequence (cos) used as a targeting vector.   FIG.Is in a transient assayCf-9(PCf9 / 10080) andCf-4(PCf4 / 10080 2) shows a binary vector for expression. Of each component See the text of the specification for details.   FIG.Is C.I. Fulbam nonpathogenic geneAvr9OrAvr4Combine with any of Under the control of the 35S promoter (35S)Cf-9OrCf-4Contains any of the genes 4 shows four binary constructs. Nonpathogenic genes are also controlled by the 35S promoter The transcription termination sequence is Claws fascence (A . tumefaciens) Octopin Provided by the intase gene 3 'untranslated region (ocs3'). Production of each plasmid Only those restriction sites that are relevant to the production (see description for details) It is shown.Positional cloning of tomato Cf-4 gene (1)Cf-4Map location   We have developed classical and RFLP maps of tomato (Dickinson et al., 1993; Jon es et al., 1993; Balint-Kurti et al., 1994; Thomas et al., 1995). Against fulbum Several genes conferring resistance were mapped. Those studies areCf-4WhenCf -9 Is bounded by two RFLP markers CP46 and TG236 on the short arm of chromosome 1. It was proved that it was located at a similar position within the defined 6 cM range.Cf-4WhenCf-9Is its Closely related speciesL . hirsutumandL . pimpinellifoliumCultured from toma (See Jones et al., 1993). Gene analysis of the present inventors May be closely linked or possibly alleles Suggest no. The present inventors have proposed a maize transposon cleavage factor ( Jones et al., 1994; transposon labeling method using PCT / GB / 02812)Cf-9Simply Released. As a result, positional cloningCf-4It became possible to isolate . (2)Cf-4Identification of gene candidates   Cf-9,Cf-4Or C. Contains unknown resistance gene to fulvum Isolate DNA from NIL of tomato and use it as restriction enzymeBglDigested with II.Cf-9of Southern hybridization analysis using the 5'-end containing probe pCf9XS (Jo nes et al., 1994) found that there was extensive RFLP among the three strains ( (Fig. 1). This result indicates that those resistance genes have been introgressed from different species That multiple members of this gene family share the same introgression chromosome segment. It was inferred from the existence on the   CandidateCf-4To identify genes, we crossed Cf4 NIL and Cf9 NILCf-4/Cf -9 Trans-heterozygotes were prepared. The offspring are then crossed with Cf0 to₁Test cross population I got About 7,500 offspring,Cf-4OrCf-9Incompatible with plants containing any of C. Fulbum variety 5 was inoculated. ifCf-4WhenCf-9Are alleles, the tiger If intragenic recombination can occur at a low frequency in parental heterozygous parents, They inferred. In some cases, this may remove the resistance specificity of each gene. Not. Progeny containing such a recombinant chromosome were identified as disease-sensitive individuals by the present inventors. Would be detected by their analysis.   Or ifCf-4WhenCf-9Are not alleles and are only closely linked , The same low frequency of intergenic recombination, which contains both resistance genes or This will result in a recombinant that does not contain both. The latter recombinant class is reduced It may also be generated as a result of chromosome mispairing or inequality during fission. The inventor In their analysis,Cf-4WhenCf-9Only progeny lacking were identified. 7,500 test crosses Among these, five such disease-susceptible recombinants were detected. Own these individuals Pollinate and the progeny again. Tested using Fulbum breed 5 and theyCf-4 WhenCf-9Make sure you have lost both. Homozygous for recombinant chromosomes Progeny were also identified. This is followed by a Southern hybridization analysis. To simplify Done forCf-9RFLP marker CP46 located 2.5 cM distal to Balint-K urti et al., 1994; Thomas et al., 1995)Cf9Individuals homozygous for the allele Was achieved by identifying   Five susceptible individuals using pCf9XS as probes (V408, V512, V514, V516 and And V517)BglII Southern blot analysis of digested DNA As expected, everything isCf-96.7 kilobase pairs (kbp) equivalent to the geneBglII Band (Joh ns et al., 1994) and lacks a number of other bands located proximal to the gene I confirmed that. Comprising different members of this gene family. do itCf-9Two separate, distally located genesBglII hand has a recombination It was present in the body but not another (FIG. 2).Cf-4Exists in the containing line ThreeBglThe II band (6.4 kbp, 3.2 kbp and 1.8 kbp, Figure 1) was also consistently affected. Lack of diseased individuals, soCf-4It was a gene candidate. This latter result is C f4 ×L.pennelliF from the cross_TwoConsistent with the results of plant analysis. TG236 / CP46 range Rearranged F_TwoThe special class of the individual is as previously described (Balint-Kurti et al. Thomas et al., 1995) identified by PCR analysis of leaf material. Those results Is threeBglII restriction fragmentCf-4It was proved to be separated with. Southern Hybrid Using the ligation and AFLP analysis, we set out the three class set shown in FIG. The variants could be distinguished. The results areCf-4WhenCf-9Recombination lacking both Consistent with models of chromosome mismatch and heterozygosity that give rise to chromosomes Does not. (3)Cf-9Isolation of binary vector cosmid clones containing homologous sequences   On chromosome 1Cf-4Genes and on chromosome 6Cf-9From a stock containing both genes, Using a library to isolate both genes A genomic DNA library was made as possible. The library is located in the UK C. of John Innes Center in Ridge. Binary cosmid obtained from Dr. Dean Made in the cloning vector pCLD04541 (see Bent et al., 1994). like that The use of cloning vectors is important for any clone It is useful because it can be introduced directly into plants for the purpose of testing gene function. It is profitable.   Using techniques well known to those skilled in the art (Thomas et al., 1994), leaves of 6-week-old greenhouse plants High molecular weight DNA fromMboPartial digestion with I restriction enzyme was performed. Techniques known to those skilled in the art Using a technique, the partially digested product is size fractionated using a sucrose gradient and DNA with a range of 20-25 kbpBam It was ligated with pCLD04541 DNA digested with HI. After packaging in vitro using Stratagene packaging extract, Erikia Kori (Escherichia coli) Stock SURE^TM(Stratagene) Tetracycline The above-mentioned cosmid was introduced into the mutated mutant. Recombinants were selected using the tetracycline resistance gene on pCLD04541.   The library was removed from 144 pools containing approximately 1500 clones per pool. Distribute randomly, grow cells from each pool, and out of 10 ml cells, 9 ml Used for bulk plasmid DNA extraction and 1 ml after addition of 0.2 ml glycerol Was used for preparation of the frozen stock. By alkali dissolution (Birnboim and Doly, 1979) Isolating the plasmid DNA from the pool and collecting the DNA sampleCf-9Contained toma Primers F7 and F10 (Jones) used to amplify the pCf9XS fragment from Et al., 1994). Pool 108 from this library, 129 and 138 were identified as pools positive for the F7 / F10 PCR product. Was. About each pool, 10,000 colonies are plated and colony hybridized using radiolabeled pCf9XS Exploration by zation. A single homologous clone was isolated from each pool. It These techniques are well known to those skilled in the art.   The three cosmid clones, designated 4 / 5-108, 4 / 5-129 and 4 / 5-138, were Southern blot hybridization using pCf9XS as a probe and restriction enzyme It was further characterized by papping. The physical relationship of the three cosmids is shown in FIG. This Analysis of 6.4, 3.2 and 1.8 kbp was consistent with the results described previously.Bgl II Restrictions It was shown that there are three regions on the fragment that show high homology with pCf9XS (FIGS. 1 and 2). And 2). These three regions were obtained from appropriate cosmid clones.PSTAs an I fragment Bucloning, using techniques well known to those skilled in the art,Cf-9To determine the genome sequence In addition to the previously used oligonucleotide primers (Jones et al., 1994), Using new primers specific for each of the three homologs, their DNA sequences It was determined. Derived from cosmids 4 / 5-108 and 4 / 5-129, respectivelyPSTI clone , The DNA sequences of homologues I and II were completely determined. Kos containing homolog III Mid 4 / 5-129PSTDNA sequence analysis of the I fragment showed that this cosmid Did not contain a complete copy of Physical mapping data is stored in this array Suggests a larger truncation on cosmid 4 / 5-138 ( (Fig. 3). This explanationCf-4/Cf-5Homolog II isolated from cDNA library It was confirmed by DNA sequence analysis of the cDNA clone of I. This analysis is A copy of homologue III on mid 4 / 5-129 is only 74 amino acids at its C-terminus Indicated that it was cut off. Homolog I (862 amino acids), Homolog II (806 amino acids) Amino acids) and homologous III (855 amino acids)Cf-9To High levels of homology (86.5%, 91.5% and 86.5%, respectively, with identical amino acids) ). (4) In plantsCf-4Development of efficient assays for gene function   Putative cloning in transgenic plantsCf-4Gene functions vary Can be evaluated in various ways. First of all, the corresponding non-pathogenic geneAvr4Including C. Inoculation of fulvum cultivars causes varieties in transgenic plants By testing whether it gives a synergistic response; second, a sum containing AVR4 Injection of intracellular fluid isolated from synergistic interactions into leaves of transgenic plants Third, previously described in studies of Cf-9 / AVR9 interaction As described (Hammond-Kosack et al., 1995), AVR4 was expressed in the form of recombinant potato virus X. It can be evaluated by communicating.   C. coding AVR4 DNA sequence of fulvum gene and after processing The amino acid sequence of the mature polypeptide has been previously reported (Joosten et al., 1994). . We have C.I. Using primers from fulbum varieties 2 and 5Avr4Gene announced The amplified sequence was amplified by PCR and the sequence encoding the proposed mature polypeptide was Fused to the DNA sequence encoding the N-terminal signal peptide of tobacco PR1a protein I combined. This is due to the intracellular location in the transgenic plant in which the gene is expressed. It will promote the targeting of AVR4 towards space. This key Mela gene (SPAvr4) As previously described (Hammond-Kosack et al., 1995)Cl a I /SalI DNA fragment (FIG. 4) of cDNA copy of potato virus X Insert it into the PVX:SPAvr4Was prepared. Infectious transcripts of this recombinant virus Obtained by in vitro transfer. All nucleic acid manipulations are performed using standard techniques well known to those skilled in the art. gave.   Control experiments were designed to test the recombinant virus in three week old tomato seedlings. In. Slight mechanical at the site of virus inoculation In contrast to the Cf0 control, which only showed signs of damage,Cf-4After inoculation (d.p. i.) On day 3, the cotyledons appeared dry and eventually fell. Cf0 plant Is a symptom observed in wild-type virus, ie, chlorosis mosaic symptom. A comparable visible symptom of viral infection was 7-10 d.p.i. It was expressed in. 4-5 d.p .i. ToCf-4In containing plants, probablyCf-9In containing plantsAvr9PVX containing For the spread of the virus as previously described in similar experiments used ( Hammond-Kosack et al., 1995), necrotic lesions were observed on younger leaves. Other features And necrotic sectors in the petiole and stem. This necrotic phenotype is whole body To 14 d.p.i. ToCf-4Most of the contained seedlings die Was. Cf0 control plants did not die but showed signs of chlorosis and vein permeabilization. Therefore, This method works on plantsCf-4Provides a fast and reliable assay for function So potentiallyCf-4Transgenic to identify individuals containing the gene Applied to plants. (Five) Cf-4Binary vector cosmid claw containing a genomic copy of the gene Identification   Cf-4/Cf-5Three bios isolated from the binary vector cosmid library Narry vector cosmid clones (4 / 5-108, 4 / 5-129 and 4 / 5-138) RekaCf-4Methods known to those skilled in the art to determine if any contain a gene Using transgenic tomatoes (Lycopersicon esclentum) And cigarettes (Nicotiana tabacum cv Petite Havana) plants (Fillatti et al., 1987; Ho rsch et al., 1985).   PVX for 12 tomato transformants:SPAvr4By inoculatingCf-4Activity is detected Transgenic plants were propagated by cuttings, as appropriate. Cosmid 4/5 Transgenic tomato plant containing -108 One thing (0/3) PVX:SPAvr4No dependent leaf necrosis was shown. In contrast, Six of eight transformants containing cosmid 4 / 5-129 and cosmid 4 / 5-138 Three of the four transformants, including 3-4 d.p.i. Leaves in inoculated leaves It showed necrosis (Table 1). This necrosisCf-4As previously observed in control plants Eventually it spread throughout the body. Most transgenic plants exhibiting the necrotic leaf sector Eventually withered. Many transgenic plants obtained from the first transformation experiment The cuttings of the object are C. By inoculation of fulvum varieties 5Cf-4Further assays for function (Table 1).   In those tests, PVX:SPAvr4Transgenic plants showing dependent necrosis Cladosporium Fulbum (Cladosporium fulvum) With those resistant to variety 5 In between, a positive correlation was observed (Table 1). Pathogen increase in compatible control plants (Cf0) Breeding was observed, but not in the incompatible control plant (Cf2). some The transgenic offspring of the transgenic plant were again Tested with fulbum variety 5 and its shape We have verified that the quality is hereditary. This has proven to be the case and in most cases The resistance was segregated at an approximately 3: 1 ratio consistent with the monodentate T-DNA transformant (Table 2). ). Cultivar 4 is not expected to express the Avr4 peptide (Joosten et al., 1994). C. Transgenic offspring resistance to fulbum cultivar 5 Did not confer resistance.   Homologue III is completely missing in cosmid 4 / 5-108 and Only truncated copies are present on the other two cosmids. Therefore, homologs III isCf-4It is not likely to be a candidate.Cf-2(Dixon et al., 199 6), multiple genes on this locus confer AVR4 peptide recognition and pathogen resistance It is possible. However, the latter example (Cf-2) Has two genes Hand Are the same, and the threeCf-9Contrast with deduced amino acid sequence of homologous gene It is a target. Complete copies of homolog I are present in all three cosmids (FIG. 3). Physical mapping and PCR determination of cosmid 4 / 5-108 was performed on this clone. Homolog II is truncated, 54 amino acids from its C-terminus and 3 ' Lack of untranslated regions and associated transcription termination and polyadenylation sequences Indicated.   Clearly, those data clearly indicate that homologue IICf-4Assume that it is related to genes. PVX :SPAvr4-Dependent leaf necrosis and C.I. The resistance to fulbum cultivar 5 was the completion of homolog II. Transgenic plants transformed with a binary vector containing the entire copy It is evident only in the product (Table 1). Multiple resistant primary forms using the pCf9XS probe Transformants (4 / 5-129A, 4 / 5-129B, 4 / 5-129D, 4 / 5-129G, 4 / 5-129H, 4 / 5-138A and And 4 / 5-138B) showed that they were all homologous II. Characteristic 3.2 kbpBglII DNA fragment.   The homolog II specifically recognizes AVR4, Sgenic cigarettes (N . tubacum) Further evidenced by the results of plant analysis. PV X:SPAvr4When inoculated (Table 3), most of the transformants containing cosmid 4 / 5-129 Most of the transformants (4/5) containing (7/10) and cosmid 4 / 5-138 had 3 to 3 4 d.p.i. At the site of virus inoculation, in several virus resistant variants It showed necrotic lesions that were identical in appearance to those seen in response to virus inoculation. Those In individuals of the group, necrosis ultimately coalesces beyond local lesions, and 7-10 d .p.i. In this case, leaf necrosis is clearly visible in all areas of the viral infection. some In transformants, PVX:SPAvr4Response is more acute and leaf necrosis sector ー 3-4 d.p.i. (Table 3). Their phenotype is cosmid 4 / 5-108 PVX also contains transgenic tobacco containing 0/5 (see Table 3):SPAvr4In cha Neither was observed in the ranged, untransformed control plants. (6) Analysis of protein encoded by homolog II   DNA sequence of homolog II (SEQ ID NO: 1, hereinafterCf-4FIG. 5). This Is 806 amino acids as shown by SEQ ID NO: 2 (FIG. 6) in concept translation. It contains a long, contiguous open reading frame encoding the acid protein. 3 'adjacent area KickCf-4WhenCf-9Nucleic acid sequence homology is very high, determined by cDNA analysis. WasCf-9Between the stop codon of the transcript (Jones et al., 1994) and the polyadenylation site They are exactly the same.   Cf-4Analysis of cDNA clones after PCR amplification derived from transcriptsCf-9 Shows that the 3 'untranslated region contains one intron as in FIG. ).   Cf-4Amino acid sequence andCf-9Comparison with the 863 amino acid sequence of (91.5% identical, 95.5% similar).Cf-9 As inCf-4The amino acid sequence was proposed by Jones et al. (1994). It has seven such putative structural domains (FIG. 7).   Domain A (amino acids 1-23) is consistent with the signal peptide sequence.   Domain B (amino acids 24-81)Cf-4This region corresponds to the mature N-terminal region of IsCf-9Includes a deletion of 10 amino acids as compared to   Domain C (amino acids 82-702) is a 26 incomplete copy of the 24 amino acid LRR sequence including. This area isCf-9Starts at LRR 10 and at LRR 12 It contains a deletion of 46 amino acids corresponding to the last two complete LRRs (FIG. 7).Cf-9WhenCf -Four Amino acid variation between It is also present in the N-terminal half of the protein (Figure 7). This area is probably related to non-disease Specifically interacts with the progenic peptide or another factor that binds to the appropriate non-pathogenic peptide It may represent a region in each protein that interacts.   In sequences C-terminal to this region, the putative Cf-4 and Cf-9 proteins are highly compatible. Amino acids 455-806 of Cf-4 and 512-863 of Cf-9 (Jones et al. 1994) is the same.   Domain D (amino acids 703-730) has no outstanding features.   Domain E (amino acids 731 to 748) contains ten negatively charged residues and contains Sex.   Domain F (amino acids 749-785) is very hydrophobic and coincides with the transmembrane domain I do.   Domain G (amino acids 786-806) is significantly basic and has eight positively charged And only two negatively charged residues.   Like Cf-9, Cf-4 protein is expected to be a membrane-bound extracytoplasmic protein. It has many of the properties that are made. (7) Tomato transgenic plant expressing AVR4 peptide   Previous work (Hammond-Kosack et al., 1994) described a transgenic expression of AVR9. Crossed with plantsCf-9Progeny of containing plants show plant death regulated during development Proved. Seedlings germinate until day 10 after germination (dpg), when necrosis is observed in cotyledons. And are phenotypically distinguishable from their wild-type counterparts. This necrosis is progressive Seedlings die at 15 dpg.   We used the SP: AVR4 cassette (FIG. 4).ClaI /BamVector as HI fragment Cloned into SLJ4K1 (Jones et al., 1992) and used in plant plants for stable expression. Motor (CaMV 35S) and transcription termination (nos3') Provided regulatory sequences. This clone was named pAVR4 / 4K1. 35S: Avr4:nos3’CassetteBglII /HinCut out as dIII fragment,BglII andHindIII Cloned into the binary transformation vector SLJ7291 (FIG. 9) digested with Thus, plasmid pAVR4 / 7291 was prepared.   Transgenic plants were prepared and tested for AVR4 expression by test crossing with the Cf4 line. Screened. Screening test hybrid offspring for seedling lethal phenotype did. 1: 1 wild-type and necrotic seedlings from crosses with 6 independent transformants The offspring to be separated were identified (AVR4 / 7291J, AVR4 / 72910, AVR4 / 7291L, AVR4 / 7291N, AVR4 / 7291M and AVR4 / 7291G).   In their progeny, the pattern of necrosis was observed in plants expressing AVR9 and CF-9. Essentially as described.   AVR4 / 7291G (female parent) andCf-4With the primary transformant (4 / 5-129H) expressing In the offspring from the miscellaneous, as expected in the cross between monodentate hemizygous plants, the offspring (wild type 35: Seedling mortality was observed in 25% of necrotic types 10). Put those seedlings in the growing room When germinated in normal agar, the pattern of necrosis differs from the control cross described above. Was. Seedlings that eventually showed necrosis were corrected and compared to their wild-type siblings, resulting in necrosis. Was evident at an earlier developmental stage. Furthermore, in plants expressing Cf-9 and AVR, An unidentified phenotype, a necrotic sector at the root, was also observed. Child of 4 / 5-129H PVX to grandchild:SPAvr4Necrosis was evident earlier than in Cf4 control plants became. This phenomenon is due to the increased trait as a result of the chromosomal location of the T-DNA insertion Transformant 4 / 5-129HCf-4May reflect expression.   This result is sufficient for non-photosynthetic tissues such as roots.Cf-4Is expressed If necrosis can be induced in the presence of AVR4 Suggests. Therefore, this result shows that in a wide range of plants, Root pathogens, such as root-dwelling bacteria or nematodes, to engineer disease resistance in tissues Use of a two-component system (eg WO 95/31564) to create resistance to Show implicitly. (8) Tomato transgenic plant containing only homolog II   Homolog II recognizes AVR4 and C. Conclusion that it is the cause of resistance to fulbum variety 5 To further investigate the theory, the high level of gene expression in A promoter capable of directing the present, ie, cauliflower mosaic virus 35 Using a homolog II under the control of the S promoter (35S; see Jones et al., 1992), A transgenic transgenic plant was produced. Their production is carried out using DNAs well known to those skilled in the art. Needed operation and modification.   Cosmid 6.0 kbp from 4 / 5-129PSTI fragment (129P1, see FIG. 3) From the car arrayEcoRI andHinIn a modified pUC119 vector lacking the daIII restriction site Cloning resulted in clone p129P6A. First, an oligonucleotide Perform otide mutagenesis to identify internal motifs beginning at nucleotides 308 and 312, respectively.Xba I andEcoThe RI restriction site was removed to create clone p129P6A-3. Such a strange Further alteration did not alter the predicted amino acid composition of the Cf-4 protein.   To express homolog II under its own promoter, p129P6A-3 to 6 .0 kbpxbaI /BamCut the HI cassette,xbaI /BamSLJ7291 digested with HI (Figure Cloning into 9) yielded clone pCf4XB / 7291. High in plants Bell'sCf-4Additional oligonucleotides may be used to create a vector that will confer expression. P129P6A-3 was engineered by ptide mutagenesis. Similarly, this change Do not change the predicted amino acid composition of the Cf-4 protein, all changes are in the DNA of the completed construct Confirmed by analysis.   These changes include the following additional modifications: (i) construct SLJ4K1 (Jones  Et al., 1992) to promote fusion to the 35S promoter.  Start with (Fig. 5)ClaIntroduction of an I restriction site (ATCGAT); (ii) beginning at nucleotide 1766 InsideHindeletion of dIII restriction site; (iii) internal beginning at nucleotide 2096BglPart II (Iv) in the 3 'untranslated region starting at nucleotide 2685ClaI restriction site Delete.   The modified homolog II coding sequence and the 3 'untranslated regionClaI /BamCut out as HI fragment And clone it into SLJ4K1 (Jones et al., 1992) to obtain plasmid p4K1 / Cf4. Produced. 35S:Cf-4CassetteBglII /Hincut it as a dIII fragmentB am HI /HinCloning into SLJ7291 cut with dIII and plasmid p35SCf4 / 7291 was produced. Tomato tran using the two binary vector clones Make sgenic plants and PVX: SPAvr4 or C.I. Fulbum variety Tested with 5 inoculations.   Several independent transformants, including pCf4XB / 7291, were inoculated with PVX: SPAvr4 It showed systemic necrosis (Table 4). Of the three transgenic plants tested, Two are C.I. It was also sufficiently resistant to fulbum variety 5. Including p35SCf4 / 7291 Eight transgenic plants show PVX: SPAvr4-dependent necrosis and some The test plant of C.I. It appeared to provide sufficient resistance to Fulbam cultivar 5 (Table 4).   These results indicate that homologue II was responsible for AVR4 recognition and C. Ten for fulbum variety 5 Prove that it is necessary and sufficient to provide adequate resistance. (9) Tobacco transgenic plant containing only homolog II   Previous experiments were observed in tomato progeny expressing Cf-9 and AVR4. It was shown that a seedling lethal phenotype could also be observed in tobacco. Cosmid 4 / 5-129 or Two subclonedPSTBinner containing any of the I fragments (FIG. 3) A Lie vector plasmid was prepared. Using those techniques known to those skilled in the art, And cloned into plasmid SLJ755A, plasmids p129P1 / 755 and p129P2 / 755 Was prepared. The binary vector SLJ755A is for Agrobacterium tumefaci Ensu (A . tumefaciens)nosStreptomyces under the control of gene promoter Su hygroscopicus (Streptomyces hygroscopicus) Contains the BAR gene. This gene, by conferring resistance to the herbicide phosphinothricin, It has been used as a transformation marker in plants (Jones et al., 1992).   These constructs worked well in tobacco transformation experiments, but not in tomatoes. Another vector was used for tomato transformation (see above).   Transgenic tobacco plants are inoculated with PVX: SPAvr4 and the binary Necrotic lesions or sectors as observed in cosmid vector transformation experiments Monitoring the emergence ofCf-4Tested for activity. p129P2 / 7 Characterize necrotic lesions and sectors among seven transgenic plants containing 55A None shown; in contrast, 13 transgenic plants containing p129P1 / 755A Ten of the products exhibited such a phenotype (Table 5). This result indicates that Homolog IICf-4 This is again consistent with the observations before adding the function.   Tobacco transgenic plants containing pAVR4 / 7291 (see above) were also generated.Cf -Four A primary transformant (as a female parent strain) containing p129P1 / 755A expressing pAVR4 / 72 Crossing with 91 transgenics, F₁Monitor seedling lethality in offspring By doing so, individuals expressing AVR4 were identified. Several transgenes expressing AVR4 Sgenics were identified (AVR4 / 7291 I, AVR4 / 7291 K and AVR4 / 7291 J) .   All F crossed₁In progeny (Table 5), both transgenics Is required for lethality in the offspring of a single T-DNA locus hemizygous parent As expected, lethality was observed in 25% of the seedlings. At the developmental stage of seedling lethality Changes in the progeny of the various crosses, at least at the macroscopic level, Not observed. However, the phenotype observed for the Cf-4 / AVR4 interaction is It differs from the phenotype observed for the Cf-9 / AVR9 interaction.   Specifically, tobacco seedling lethality is observed at an early stage of seedling development. (Normal agar At day 7 after seeding, wild-type sibs are readily identified as expressing Cf-4 / AVR4 Can be distinguished. Seedling cotyledons expressing Cf-4 and AVR4 did not completely elongate, Does not produce appreciable amounts of chlorophyll. Most seedlings have one seed coat It remains attached to the cotyledon or surrounds the two cotyledons. The latter No features were observed in tobacco seedlings expressing Cf-9 / AVR9. 14 days after sowing Seedlings expressing Cf-4 / AVR4 appear completely necrotic. Those Seedlings develop a broad root system as reported for the Cf-9 / AVR9 interaction I can't do that either.   These results indicate earlier developmental stages than those reported for the Cf-9 / AVR9 interaction. Suggests that seedlings will appear on the floor. (10) Transient expression assay to monitor Cf-4 and Cf-9 activity   Experiments in our laboratory focus on binary vectors under the control of the CaMV 35S promoter. The gene on the plasmid is Claws fascence (A . tumefaciens) Fungus When introduced into the leaves by infiltrating the suspension, tobacco (Nicotiana t abacum ) Demonstrated that it can be transiently expressed in leaf cells. AVR9 expression Regular transgenic 35S:Cf-9In experiments using infiltrated leaf panels, Necrosis was observed. This phenotype was specific for plants expressing AVR9 .uidA(GUS) Another experiment using intron-containing variants of the reporter gene is It was proved that this phenomenon was a result of nuclear gene expression in plants. We use it for cigarettes The method used was kidney beans (Phaseolus vulgaris) Transient gene expression in leaves It was a variant of the protocol developed for the assay.   Binary vector plasmids are cloned by triple parenting, a technique well known to those skilled in the art. A. Transferred into Tumefaciens GV3101 / pMP90. Antibiotic rifampicillin (50 μg / ml) and L-broth medium containing tetracycline (1 μg / ml) A single colony was picked and placed on a 5 ml L broth containing antibiotics in a shaking incubator. Grow at 48 ° C for 48 hours. One milliliter of this saturated culture was made with 10 mM 2- (N -Morpholino) ethanesulfonic acid (MES) pH 5.6 and Agrobacterium (Agrobacterium )VirContains 20 μM acetosyringone to induce the gene L broth in 100 ml. Incubate the culture at 28 ° C for 16 hours in a shaking incubator. Was. Bacterial cells are pelleted by low-speed centrifugation, Murashige & Skoog (MS) salt, 2% w / v sucrose, containing 500 μM MES pH 5.6 and 10 μM acetosyringone Resuspended in buffer. OD of each culture₆₀₀And OD₆₀₀= 0.5 Was adjusted as follows. The culture was then incubated at 22 ° C. without shaking for 3 hours.   tobacco(N . tabacumMake several small cuts in the target leaf panel on the adult leaf After that, the Agrobacterium suspension was infiltrated with a syringe. Number of panels After injection, the infiltrated leaves were covered with a plastic bag to prevent drying. 72 hours later, Removed.   35S:Cf-4Similar experiments using Agrobacterium containing binary vectors That express AVR4 constitutively (non-regulated) Ko (N . tabacum) Leaves infiltrated. 35S:Cf-4The binary vector is as follows It was produced.   E. FIG. From KoriuidA1.8 kbp (GUS) geneClaI /BamHI fragment Plasmid p4K1 / Cf4 in the binary vector SLJ10080 (see item (8)) ) Of originCf-4Codes for 2.9 kbpClaI /BamReplaced with HI fragment.Cf-9DNA Similar changes, including oligonucleotide mutagenesis, performed as above and similar Of the composition. Obtained binary vector plasmid pCf4 / 10080 and pCf4 / 10080 f9 / 10080 to A. Populated during the claws.   In transient expression assays, assays containing pCf4 / 10080 or pCf9 / 10080 Any controls without the bacterium suspension or the binary vector It did not induce any visible necrosis in untransformed tobacco. pCf9 / 10080 Only infiltrated leaf panels in tobacco expressing AVR9 5-6 days after infiltration Induced a necrotic response. Transgenic plant AVR4 / 7291 K (see item (9) ), Only leaf panels infected with pCf4 / 10080 were visible 5 to 6 days after infiltration. Induced visible necrosis.   Therefore, this assay may beCf-9WhenCf-4Test for both gene function To mutateCfCreating stable transgenic plants that express genes Provide a quick and reliable way to determine the effects of a particular mutation without the need to Offer.   We also found that 35S:Cf-4Or 35S:Cf-9Remains 35S corresponding to the gene construct:AvrVector carrying both gene constructs Inferred that it could cause visible necrosis in the infiltrated leaves. If If not, the assay will generate either a resistance gene or a non-pathogenic gene component. Without the need to create transgenic plants to showCf-4andCf-9Works Methods can be provided for determining the range of species that can be used. this is To confer resistance in different crop speciesCfGene / Non-pathogenic gene two-component system (WO 95/31564) will have important implications.   Binary vector plasmids containing both resistance and non-pathogenic genes It was produced as follows. 35S:Cf-4And 35S:Cf-9Insert the cassette into pCf4 / 10080 and And pCf9 / 10080 were excised as follows: (i)Apa Digest with T1 and T4 DNA (Ii) subjecting the DNA fragments to blunt ends by treatment with a lase;PST35S digested with I:Cf-4You And 35S:Cf-9Releasing the cassette; (iii) vector SLJ456 (Jones et al., 1992) ToClaLinearize with I and treat with T4 DNA polymerase to make the DNA blunt-ended And thenPSTI digested with 2.1 kbp neomycin phosphotransferase ( NPT) to release the reporter gene fragment; (iv) 4.3 kbp 35S:Cf-4And 4.5  kbp 35S:Cf-9 ApaI (T4) /PSTI fragmentClaI (T4) /PSTI processed Cloning into SLJ456 created plasmids p4 / 456 and p9 / 456. This All of these techniques are well known to those skilled in the art.   Nonpathogenic gene constructs were made as follows.   Clone pAVR4 / 4K1 (see item (9))EcoRI andBam35S digested with HI : The AVR4 cassette was excised. The purified fragment was purified from SLJ6B1 (Jones et al., 1992). About 3.9 kbpEcoRI /BamLigation with the HI fragment, 35S, AVR4 and A. Tsumefa Science (A . tumefaciens) Transcription terminator of octopine synthase gene (ocs3') Containing the plasmid pAVR4 / 6B1. Clone SLJ6071 (Ham From mond-Kosack et al., 1994), 35S: AVR0:ocs3'' Make the component Thus, a plasmid pAVR9 / 6B1 was obtained. Plasmids pAVR4 / 6B1 and pAVR9 / 6B1HindIII And linearize with T4 DNA polymerase. The DNA fragment was blunt-ended. 35S: AVR4:ocs3'And 35S: AVR9:oc s3 '' CassetteBglIsolated by treatment with II. Each purified cassetteBamHI /Hpa  Ligated with p4 / 456 and p9 / 456 treated with I Each resistance gene (in combination with anyCf-4OrCf-9Assay the function of Four types of plasmids were prepared.   tobacco(Nicotiana benthamiana) Four leaf panels on a single leaf of the plant Agrobacterium containing each of the four plasmids shownAgrobacteriumDipped) Moisturized. 5 days after infiltration in panels injected with vector p4 / 456 / AVR4 Death was observed. No necrosis was observed in the panel injected with p4 / 456 / AVR9. this is We show that the latter result is due to co-expression of Cf-4 and AVR4 in the same cells.   After 7 days, the necrotic leaves were removed and stored at room temperature in sealed polyethylene bags. Finally, necrosis was observed 12 days after infiltration in the leaf panel injected with p9 / 456 / Avr9 , And was not observed in leaf panels injected with p9 / 456 / Avr4.   These experiments use different species when cognate non-pathogenic determinants are co-expressed. In a transient expression assayCfProven ability to detect necrosis-inducing activity of genes I do. Necrosis induced by the Cf-4 / AVR4 interaction is due to the Cf-9 / AVR9 interaction. Appeared much earlier than triggered. Promoter, translation and This phenomenon is due to their corresponding non-pathogenic It may reflect the difference in the relative affinities of Cf-4 and Cf-9 for the gene product. This The theory is that transgenic tobacco expressing Cf-4 and AVR4 emits Cf-9 and AVR9. This is consistent with the emergence of a seedling lethal phenotype earlier than that which appears.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] June 6, 1997 [Correction contents]   9. 9. The method of claim 8, further comprising a regulatory sequence for expression of the polypeptide. A nucleic acid as described.   Ten. In the production of transgenic plants, according to any one of claims 1 to 9 Use of the described nucleic acid.   11． A host cell comprising the exogenous nucleic acid according to any one of claims 1 to 9.   12． 12. The host cell according to claim 11, which is a microbial cell.   13. 12. The host cell according to claim 11, which is a plant cell.   14. A plant comprising a cell according to claim 13 or any part thereof.   15． A seed, inbred or hybrid progeny or progeny, derivative of the plant of claim 14. Or extract, or any part thereof.   16． A method for introducing the nucleic acid according to any one of claims 1 to 9 into a host cell. Comprising a method.

──────────────────────────────────────────────────続 き Continuation of front page (51) Int.Cl. ⁶ Identification symbol FI A01N 65/00 C12N 5/00 C (C12N 1/21 C12R 1:19) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, P, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Ballint-Coty, Peter John United States of America, Maryland 20892, Bethesda, Room 1-22, Building 6, Laboratory of Cellular Developmental Biology, National Institute of Diabetes and Degestive and Kidney Disuse, National Institute of Health (72) Inventor Jones, David Allen United Kingdom, Norwich N. 46 P.D., Eaton, Greenways 139 (72) Inventor Jones Jonathan Dallas Joe di UK, Norfolk Enuaru 4 6 ISG, Norwich, Waverly Road 19

Claims (1)

[Claims]   1. Pathogens whose expression in plants can trigger activation of plant defense responses A nucleic acid isolate encoding a body resistance gene, comprising the amino acid set forth in SEQ ID NO: 2 Nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the sequence Isolate.   2. Said activation is due to contact of the pathogen or the corresponding elicitor molecule with the plant The nucleic acid of claim 1.   3. The nucleotide sequence comprises the coding sequence shown in SEQ ID NO: 1. The nucleic acid according to claim 1.   4. The nucleotide sequence is an addition or insertion of one or more nucleotides, Alleles or derivatives of the coding sequence shown in SEQ ID NO: 1 due to deletion or substitution Or the nucleic acid of claim 1, comprising a variant.   5. Pathogens whose expression in plants can trigger activation of plant defense responses A nucleic acid encoding a body resistance gene, wherein the nucleic acid encodes a polypeptide. Wherein the polypeptide comprises one or more amino acids, An allele of the amino acid sequence shown in SEQ ID NO: 2 by insertion, deletion or substitution, Comprising an amino acid sequence comprising a derivative or variant,   However, the encoded polypeptide differs from the amino acid sequence shown in SEQ ID NO: 2 by a small amount. A nucleic acid isolate premised on having at least about 95% amino acid sequence identity.   6. Pathogens whose expression in plants can trigger activation of plant defense responses A nucleic acid encoding a body resistance gene, wherein the nucleic acid encodes a polypeptide. Wherein the polypeptide comprises one or more amino acids, By insertion, deletion or substitution, Including alleles, derivatives or variants of the amino acid sequence shown in SEQ ID NO: 2 Comprising an amino acid sequence consisting of   However, the expression of the nucleic acid is protected by contact between the plant and the Avr4 elicitor molecule. Nucleic acids that are premised on being able to cause a responsive activation.   7. Said activation is due to contact of the pathogen or the corresponding elicitor molecule with the plant The nucleic acid according to claim 5 or 6.   8. A nucleus, which is a vector comprising the nucleic acid according to any one of claims 1 to 7. acid.   9. 9. The method of claim 8, further comprising a regulatory sequence for expression of the polypeptide. A nucleic acid as described.   Ten. In the production of transgenic plants, according to any one of claims 1 to 9 Use of the described nucleic acid.   11． A host cell comprising the nucleic acid according to any one of claims 1 to 9.   12． 12. The host cell according to claim 11, which is a microbial cell.   13. 12. The host cell according to claim 11, which is a plant cell.   14. A plant comprising a cell according to claim 13 or any part thereof.   15． A seed, inbred or hybrid progeny or progeny, derivative of the plant of claim 14. Or extract, or any part thereof.   16． A method for introducing the nucleic acid according to any one of claims 1 to 9 into a host cell. Comprising a method.   17． 17. The method of claim 16, wherein the host cell is a plant cell or a microbial cell. .   18． A method for conferring pathogen resistance on a plant, comprising the plant or its ancestors Following the initial stage of introducing the nucleic acid into cells of the plant, Comprising expression from a nucleic acid according to any one of claims 1 to 9 in a cell of Method.   19. The method according to claim 18, wherein the nucleic acid encodes the amino acid sequence shown in SEQ ID NO: 2. The described method.